The present invention, in some embodiments thereof, relates to methods of generating embryoid bodies and to cell cultures comprising same.
Human embryonic stem cells (hESCs) are pluripotent cells that can potentially differentiate into all cell types present in the adult body. However, the promise of hESCs in providing unlimited supply of cells for therapy greatly depends on the availability of controllable large-scale bioprocesses. Potential industrial applications will require a large number of cells, thus enhancing the need to develop scalable methods for the production and differentiation of hESCs. The culture volumes of few milliliters utilized in basic biology must be adapted to clinically relevant scales of up to hundreds of liters.
In-vitro ESCs differentiation commonly requires spontaneous formation in suspension of spherical cell clusters called embryoid bodies (EBs). Typically, EBs include cells derivatives of all three primary germ layers—endoderm, mesoderm and ectoderm. It is known that mouse EBs can be generated from a single mouse ESC. In contrast, due to the low clonality of human ESCs, formation of human EBs requires initial ESCs aggregation while preventing EBs' agglomeration, which may affect cell proliferation and differentiation and cause extensive cell death (Dang S M., et al., 2002; Schroeder M., et al., 2005; Xu et al., 2002; Dang et al., 2004). Standard methods of generating EBs include hanging drop, liquid suspension and methylcellulose culture. However, these methods are not efficient for the industrial arena due to their complexity.
Direct seeding of mESCs into a spinner flask equipped with a paddle-impeller resulted in the formation of large ESC clumping and agglomeration within 3 days (Schroeder M., et al., 2005). Attempts to avoid agglomeration include increase of stirring rate (Chisti, 2001); encapsulation of mESCs prior to seeding in the stirred culture (U.S. Pat. Appl. No. 20030119107 to Dang and Zandstra); pre-incubation of mouse EBs in Petri dishes prior to their transfer into paddle impeller spinner flasks (Zandstra et al., 2003); spinning of Petri dishes on a horizontal rotation device (Zweigerdt et al., 2003); direct seeding of mESCs into spinner flasks equipped with a glass bulb-shaped impeller or into a 2-liter vessel equipped with a pitched-blade turbine impeller (Schroeder et al., 2005) or direct seeding of mESCs into Fernbach flask on a rotary shaker (WO 04039966 to Gryseels T D et al.).
Cameron C M., et al., 2006, describe formation of hEBs by cultivation of human ESCs in a spinner flask which contains a magnetic stir bar.
U.S. Pat. Appl. No. 20060148078 and Gerecht-Nir et al. 2004 describe dynamic generation of human embryoid bodies in bioreactors such as the Rotating Wall Vessel (RWV) bioreactor (known also as Slow Turning Lateral Vessel—STLV).
Additional background art includes Wartenberg et al., 1998; U.S. Pat. Appl. No. 20040096967; U.S. Pat. Appl. No. 20060134782.